UMR3244 – Dynamique de l’information génétique

Publications de l’équipe

Année de publication : 2009

Julia Berretta, Antonin Morillon (2009 Aug 15)

Pervasive transcription constitutes a new level of eukaryotic genome regulation.

EMBO reports : 973-82 : DOI : 10.1038/embor.2009.181 En savoir plus
Résumé

During the past few years, it has become increasingly evident that the expression of eukaryotic genomes is far more complex than had been previously noted. The idea that the transcriptome is derived exclusively from protein-coding genes and some specific non-coding RNAs–such as snRNAs, snoRNAs, tRNAs or rRNAs–has been swept away by numerous studies indicating that RNA polymerase II can be found at almost any genomic location. Pervasive transcription is widespread and, far from being a futile process, has a crucial role in controlling gene expression and genomic plasticity. Here, we review recent findings that point to cryptic transcription as a fundamental component of the regulation of eukaryotic genomes.

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Valérie Borde, Nicolas Robine, Waka Lin, Sandrine Bonfils, Vincent Géli, Alain Nicolas (2009 Aug 6)

Histone H3 lysine 4 trimethylation marks meiotic recombination initiation sites.

The EMBO journal : 99-111 : DOI : 10.1038/emboj.2008.257 En savoir plus
Résumé

The function of histone modifications in initiating and regulating the chromosomal events of the meiotic prophase remains poorly understood. In Saccharomyces cerevisiae, we examined the genome-wide localization of histone H3 lysine 4 trimethylation (H3K4me3) along meiosis and its relationship to gene expression and position of the programmed double-strand breaks (DSBs) that initiate interhomologue recombination, essential to yield viable haploid gametes. We find that the level of H3K4me3 is constitutively higher close to DSB sites, independently of local gene expression levels. Without Set1, the H3K4 methylase, 84% of the DSB sites exhibit a severely reduced DSB frequency, the reduction being quantitatively correlated with the local level of H3K4me3 in wild-type cells. Further, we show that this differential histone mark is already established in vegetative cells, being higher in DSB-prone regions than in regions with no or little DSB. Taken together, our results demonstrate that H3K4me3 is a prominent and preexisting mark of active meiotic recombination initiation sites. Novel perspectives to dissect the various layers of the controls of meiotic DSB formation are discussed.

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Marina Pinskaya, Antonin Morillon (2009 Jul 28)

Histone H3 lysine 4 di-methylation: a novel mark for transcriptional fidelity?

Epigenetics : 302-6 En savoir plus
Résumé

Although histone H3 Lysine 4 methylation (H3K4me) is strongly associated with active transcription, an increasing number of arguments indicate its repressive role in gene expression. Recent data in the mammalian and budding yeast systems have provided evidence for H3K4me2 and H3K4me3 tethering histone deacetylase complexes (HDACs) to modulate gene expression. In S. cerevisiae, this regulation is mediated by specific subunits within HDACs that recognize the methylation status of H3K4 allowing chromatin reorganization to attenuate or repress transcription. Albeit we are still a long way from understanding the mechanism and biological consequences, it is becoming clear that H3K4me at certain chromatin loci may prevent aberrant gene expression or modulate transcriptional response. This review will provide a brief overview of a novel interpretation of H3K4me and its outcome on transcription regulation and will suggest future challenges for the field of epigenetics.

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Franck Toledo, Boris Bardot (2009 Jul 24)

Cancer: Three birds with one stone.

Nature : 466-7 : DOI : 10.1038/460466a En savoir plus
Résumé

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Marina Pinskaya, Stéphanie Gourvennec, Antonin Morillon (2009 May 2)

H3 lysine 4 di- and tri-methylation deposited by cryptic transcription attenuates promoter activation.

The EMBO journal : 1697-707 : DOI : 10.1038/emboj.2009.108 En savoir plus
Résumé

Set1-dependent H3K4 di- and tri-methylation (H3K4me2/3) have been associated with active transcription. Recent data indicate that the H3K4me2/3 also plays a poorly characterized RNA-dependent repressive role. Here, we show that GAL1 promoter is attenuated by the H3K4me2/3 deposited by cryptic transcription. The H3K4me2/3 delay the recruitment of RNA polymerase II (RNAPII) and TBP on GAL1 promoter. Inactivation of RNA decay components revealed the existence of the RNAPII-dependent unstable RNAs, initiating upstream of GAL1 (GAL1ucut). GAL1ucut RNAs are synthesized in glucose and require the Reb1 transcription factor. Consistent with a regulatory function of the cryptic transcription, Reb1 depletion leads to a decrease of H3K4me3 on GAL10-GAL1 locus in glucose and to an acceleration of GAL1 induction. A candidate approach shows that the RPD3 histone deacetylase attenuates GAL1 induction and is tethered at the GAL10-GAL1 locus by H3K4me2/3 upon repression. Strikingly, Set1-dependent Rpd3 recruitment represses also the usage of a hidden promoter within SUC2, suggesting a general function for H3K4me2/3 in promoter fidelity. Our data support a model wherein certain promoters are embedded in a repressive chromatin controlled by cryptic transcription.

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Marina Pinskaya, Anitha Nair, David Clynes, Antonin Morillon, Jane Mellor (2009 Mar 11)

Nucleosome remodeling and transcriptional repression are distinct functions of Isw1 in Saccharomyces cerevisiae.

Molecular and cellular biology : 2419-30 : DOI : 10.1128/MCB.01050-08 En savoir plus
Résumé

The SANT domain is a nucleosome recognition module found in transcriptional regulatory proteins, including chromatin-modifying enzymes. It shows high functional degeneracy between species, varying in sequence and copy number. Here, we investigate functions in vivo associated with two SANT motifs, SANT and SLIDE, in the Saccharomyces cerevisiae Isw1 chromatin-remodeling ATPase. We show that differences in the primary structures of the SANT and SLIDE domains in yeast and Drosophila melanogaster reflect their different functions. In yeast, the SLIDE domain is required for histone interactions, while this is a function of the SANT domain in flies. In yeast, both motifs are required for optimal association with chromatin and for formation of the Isw1b complex (Isw1, Ioc2, and Ioc4). Moreover, nucleosome remodeling at the MET16 locus is defective in strains lacking the SANT or SLIDE domain. In contrast, the SANT domain is dispensable for the interaction between Isw1 and Ioc3 in the Isw1a complex. We show that, although defective in nucleosome remodeling, Isw1 lacking the SANT domain is able to repress transcription initiation at the MET16 promoter. Thus, chromatin remodeling and transcriptional repression are distinct activities of Isw1.

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G Tilman, A Loriot, A Van Beneden, N Arnoult, J A Londoño-Vallejo, C De Smet, A Decottignies (2009 Mar 3)

Subtelomeric DNA hypomethylation is not required for telomeric sister chromatid exchanges in ALT cells.

Oncogene : 1682-93 : DOI : 10.1038/onc.2009.23 En savoir plus
Résumé

Most human tumor cells acquire immortality by activating the expression of telomerase, a ribonucleoprotein that maintains stable telomere lengths at chromosome ends throughout cell divisions. Other tumors use an alternative mechanism of telomere lengthening (ALT), characterized by high frequencies of telomeric sister chromatid exchanges (T-SCEs). Mechanisms of ALT activation are still poorly understood, but recent studies suggest that DNA hypomethylation of chromosome ends might contribute to the process by facilitating T-SCEs. Here, we show that ALT/T-SCE(high) tumor cells display low DNA-methylation levels at the D4Z4 and DNF92 subtelomeric sequences. Surprisingly, however, the same sequences retained high methylation levels in ALT/T-SCE(high) SV40-immortalized fibroblasts. Moreover, T-SCE rates were efficiently reduced by ectopic expression of active telomerase in ALT tumor cells, even though subtelomeric sequences remained hypomethylated. We also show that hypomethylation of subtelomeric sequences in ALT tumor cells is correlated with genome-wide hypomethylation of Alu repeats and pericentromeric Sat2 DNA sequences. Overall, this study suggests that, although subtelomeric DNA hypomethylation is often coincident with the ALT process in human tumor cells, it is not required for T-SCE.

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N Arnoult, K Shin-Ya, J A Londoño-Vallejo (2009 Feb 4)

Studying telomere replication by Q-CO-FISH: the effect of telomestatin, a potent G-quadruplex ligand.

Cytogenetic and genome research : 229-36 : DOI : 10.1159/000167808 En savoir plus
Résumé

Telomere replication is a critical process for preserving genome integrity. The telomere replication fork proceeds unidirectionally from the last subtelomeric origin towards the end of the chromosome, replicating the 5′-3′ G-rich strand by lagging mechanisms and the complementary C-rich strand by leading mechanisms. It has been proposed that the G-rich nature of telomeres may favor the formation of secondary structures such as G-quadruplexes during replication and that specific mechanisms must prevent this to allow the fork to progress unimpeded. The potential of G-quadruplex formation by telomeric sequences has been clearly demonstrated in vitro but it is not known whether these structures form in vivo. We tested the effect of a potent and specific G-quadruplex ligand, telomestatin (TMS), on telomere replication using a novel quantitative approach applied to CO-FISH. We show that TMS, although it penetrates and persists within cells, does not affect telomere replication after short or long-term treatments of mouse embryonic fibroblasts. It does however affect the hybridization efficiency of FISH telomeric probes that recognize the G-rich strand. Our work illustrates the use of a novel technique to measure telomere replication efficiency and suggests that G-quadruplex ligands do not affect telomere replication in a non tumoral context.

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Valérie Borde, Jennifer Cobb (2009 Jan 20)

Double functions for the Mre11 complex during DNA double-strand break repair and replication.

The international journal of biochemistry & cell biology : 1249-53 : DOI : 10.1016/j.biocel.2008.12.013 En savoir plus
Résumé

Defining the factors that lead to genomic instability is one of the most important fields in cancer biology. DNA damage can arise from exogenous sources or as a result of normal cellular metabolism. Regardless of the cause, when damaged DNA is not properly repaired the genome acquires mutation(s). Under normal circumstances, to prevent such chromosome instability the cell activates the checkpoint response, which inhibits cell cycle progression until DNA repair is complete. The Mre11 complex is formed by three components: Mre11, Rad50, and Nbs1/Xrs2 and is involved in the signaling pathways that lead to both checkpoint activation and DNA repair. In response to DNA damage two functions of the complex will be discussed, one involves its role in initiating kinase activation and the second involves its ability to tether and link DNA strands. This review will highlight the functions of the Mre11 complex during the process of DNA double strand break recognition and repair, and during the process of replication. Understanding how the Mre11 complex is working at the molecular level is important for understanding why disruptions in components of the complex lead to genomic instability and cancer predisposition syndromes in humans.

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Année de publication : 2008

Delphine T Marie-Egyptienne, Marie Eve Brault, Graeme A M Nimmo, J Arturo Londoño-Vallejo, Chantal Autexier (2008 Dec 6)

Growth defects in mouse telomerase RNA-deficient cells expressing a template-mutated mouse telomerase RNA.

Cancer letters : 266-76 : DOI : 10.1016/j.canlet.2008.10.027 En savoir plus
Résumé

Cellular viability requires telomere maintenance, which, in mammals, is mainly mediated by the reverse transcriptase telomerase. Telomerase core components are a catalytic subunit TERT and an RNA subunit TR (hTR in humans, mTR in mouse) that carries the template to generate telomeres de novo. Telomere dysfunction can lead to senescence or apoptosis and impairs the continued growth of immortal cancerous cell lines. The introduction of a template-mutated hTR in telomerase-positive and telomerase-negative human cell lines results in dramatic growth defects. No study has addressed the consequences of expressing a template-mutated mTR in mouse immortal cell lines. Therefore, we analyzed the effects of long-term expression of a template-mutated mTR in the telomerase-positive and telomerase-negative murine cell lines CB17 and DKO301, respectively. Whereas the CB17 clones expressing the template-mutated mTR did not demonstrate any growth impairment, many of the DKO301 clones expressing the template-mutated mTR underwent growth and cell cycle defects and eventual cell death. These results suggest that in the absence of wild-type telomerase, the expression of the template-mutated mTR likely perturbs telomere function, leading to decreased cellular viability. Furthermore, whereas the expression of template-mutated hTR in telomerase-negative human cell lines leads to immediate cellular toxicity, the expression of the template-mutated mTR in the telomerase-negative mouse cell line did not.

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J Arturo Londono-Vallejo (2008 Jul 9)

[Cancer as a microevolutionary process affecting telomere structure and dynamics: the contribution of telomeres to cancer].

Ai zheng = Aizheng = Chinese journal of cancer : 775-83 En savoir plus
Résumé

Telomeres play fundamental roles in genome stability, nuclear architecture and chromosome pairing during meiosis. They shorten at every cell division and may be re-elongated or not depending on the presence of the dedicated enzyme, telomerase. Since in most human somatic cells telomerase is not expressed, shortening of telomeres during development and aging is the rule. Short telomeres being, under physiological conditions, incompatible with extended cell proliferation, telomere length defines the proliferation potential of a cell and operates as a mechanism to prevent uncontrolled cell growth. Conversely, in cells in which proliferation checkpoints have been abolished, shortening of telomeres causes chromosomes to fuse and to initiate cycles of breakage-fusion-bridge thus becoming a strong driving force for genome instability. In vitro, transformed cells with highly unstable genomes because of severe telomere shortening accumulate deleterious genetic changes and die (crisis). At the same time, random genetic or epigenetic changes may allow cells to acquire a telomere maintenance mechanism (as well as other tumor phenotypes) and to become immortal. Although telomere shortening and other types of telomere dysfunction probably contribute to the genome instability detected in early tumors in vivo, the direct contributions of dysfunctional telomeres to the acquisition of tumor phenotypes in humans remain largely unspecified.

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Arturo Londoño-Vallejo, Christelle Lenain, Eric Gilson (2008 Apr 15)

[Targeting telomeres to enforce cancer cells to senesce].

Médecine sciences : M/S : 383-9 : DOI : 10.1051/medsci/2008244383 En savoir plus
Résumé

The telomeres protect the end of chromosomes from being recognized and processed as an accidental double stranded break. In human somatic cells, telomeres shorten progressively with every round of DNA replication, leading to dysfunctional telomeres that trigger cellular senescence or apoptosis depending on the cell type. This telomere erosion appears to play a role in cell renewal, ageing and cancer. Two recent studies demonstrated in mouse that eroded telomeres in cancer cells blocked for apoptosis limit cancer formation by triggering senescence. These results suggest that provoking senescence may provide a way to cure cancer and point to new therapeutical strategies targeting specific telomeric functions. Nevertheless, an important question remains unanswered: does replicative senescence limit tumor formation in human?

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Julia Berretta, Marina Pinskaya, Antonin Morillon (2008 Mar 5)

A cryptic unstable transcript mediates transcriptional trans-silencing of the Ty1 retrotransposon in S. cerevisiae.

Genes & development : 615-26 : DOI : 10.1101/gad.458008 En savoir plus
Résumé

Cryptic unstable transcripts (CUTs) are synthesized from intra- and intergenic regions in Saccharomyces cerevisiae and are rapidly degraded by RNA surveillance pathways, but their function(s) remain(s) elusive. Here, we show that an antisense TY1 CUT, starting within the Ty1 retrotransposon and encompassing the promoter 5′ long terminal repeat (LTR), mediates RNA-dependent gene silencing and represses Ty1 mobility. We show that the Ty1 regulatory RNA is synthesized by RNA polymerase II, polyadenylated, and destabilized by the cytoplasmic 5′ RNA degradation pathway. Moreover, the Ty1 regulatory RNA represses Ty1 transcription and transposition in trans by acting on the de novo transcribed TY1 RNA. Consistent with a transcriptional regulation mechanism, we show that RNA polymerase II occupancy is reduced on the Ty1 chromatin upon silencing, although TBP binding remains unchanged. Furthermore, the Ty1 silencing is partially mediated by histone deacetylation and requires Set1-dependent histone methylation, pointing out an analogy with heterochromatin gene silencing. Our results show the first example of an RNA-dependent gene trans-silencing mediated by epigenetic marks in S. cerevisiae.

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Ulrich Schlecht, Ionas Erb, Philippe Demougin, Nicolas Robine, Valérie Borde, Erik van Nimwegen, Alain Nicolas, Michael Primig (2008 Feb 29)

Genome-wide expression profiling, in vivo DNA binding analysis, and probabilistic motif prediction reveal novel Abf1 target genes during fermentation, respiration, and sporulation in yeast.

Molecular biology of the cell : 2193-207 : DOI : 10.1091/mbc.E07-12-1242 En savoir plus
Résumé

The autonomously replicating sequence binding factor 1 (Abf1) was initially identified as an essential DNA replication factor and later shown to be a component of the regulatory network controlling mitotic and meiotic cell cycle progression in budding yeast. The protein is thought to exert its functions via specific interaction with its target site as part of distinct protein complexes, but its roles during mitotic growth and meiotic development are only partially understood. Here, we report a comprehensive approach aiming at the identification of direct Abf1-target genes expressed during fermentation, respiration, and sporulation. Computational prediction of the protein’s target sites was integrated with a genome-wide DNA binding assay in growing and sporulating cells. The resulting data were combined with the output of expression profiling studies using wild-type versus temperature-sensitive alleles. This work identified 434 protein-coding loci as being transcriptionally dependent on Abf1. More than 60% of their putative promoter regions contained a computationally predicted Abf1 binding site and/or were bound by Abf1 in vivo, identifying them as direct targets. The present study revealed numerous loci previously unknown to be under Abf1 control, and it yielded evidence for the protein’s variable DNA binding pattern during mitotic growth and meiotic development.

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Année de publication : 2007

Rebecca Johnson, Valérie Borde, Matthew J Neale, Anna Bishop-Bailey, Matthew North, Sheila Harris, Alain Nicolas, Alastair S H Goldman (2007 Dec 18)

Excess single-stranded DNA inhibits meiotic double-strand break repair.

PLoS genetics : e223 En savoir plus
Résumé

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.

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